The project aims to isolate stem cells from human salivary glands and to determine the optimal drug and growth factor combination to increase stem cell enrichment and facilitate their engraftment and differentiation in irradiated mouse submandibular (SMG) glands. Our ultimate goal is to employ human salivary stem cells to rescue salivary function in irradiated head and neck cancer (HNC) patients. Most patients with HNC require radiation therapy (RT) to manage their disease. In >70% of these patients, RT results in chronic xerostomia (dry mouth), which can adversely impact their quality of life and lead to severe subsequent complications, such as mandibular osteoradionecrosis and/or dysphagia. Current pharmacologic management of RT-related xerostomia is costly and ineffective. Intensity modulated radiotherapy (IMRT), which aims to spare one parotid gland, has resulted in some improvement of stimulatory salivary function, such as with eating. However, IMRT cannot spare the SMG glands, which are crucial for resting salivary function throughout the day, because of their locations adjacent to the draining lymph nodes. For this reason, there is strong interest in rescuing SMG function. Present efforts using induced pluripotent stem (IPS) cells or tissue engineering appear promising but are still at a very early stage. Recently published data indicate that stem cells can be isolated from adult mouse SMG glands and can form functional subunits when transplanted into irradiated recipient mice. Preliminary data also suggest that stem cells could improve saliva production in these mice. Therefore, it is desirable to test the feasibility of isolating human SMG stem cells and their ability to reconstitute functional subunits of the gland. However, these adult SMG stem cells are rare, making up only 0.3% of the entire SMG cell population. Efforts are needed to improve their isolation, survival in culture and engraftment in vivo. Aldehyde dehydrogenase isozymes (ALDH) are well-known stem cell markers whose activity levels correlate with the ability of progenitor cells to differentiate into target tissues. Recently, we found certain ALDH enzymes are expressed at high level in adult SMG stem cells compared to non-stem cells. We have identified several small molecular activators of different ALDH isozymes using a high-throughput screen. We hypothesize that activation of selective ALDH isozymes, in combination with growth factors, will enhance isolation and survival of salivary stem cells and facilitate their engraftment in vivo. We propose to (1) identify ALDH isozymes that are preferentially expressed and active in human and mouse SMG stem cells, (2) determine the optimal ALDH activator(s) to enhance stem cell isolation, survival and proliferation, (3) evaluate the efficacy of these cells to form functional subunits & rescue function in SMG of nude mice, (4) identify the minimal number of stem cells required for functional rescue, and (5) determine the optimal route and schedule of administration of ALDH activators to facilitate stem cell engraftment. These studies will support future use of SMG stem cells for salivary function restoration in irradiated HNC patients.